Ebright, R.H.; Gunasekeram, A.

Proc. Natl. Acad. Sci. U. S. A. 1990, 87, 2882-2886, 10.1073/pnas.87.8.2882

Escherichia coli catabolite gene activator protein (CAP) is a helix-turn-helix motif sequence-specific DNA binding protein [de Crombrugghe, B., Busby, S. & Buc, H. (1984) Science 224, 831-838; and Pabo, C. & Sauer, R. (1984) Annu. Rev. Biochem. 53, 293-321]. In this work, CAP has been converted into a site-specific DNA cleavage agent by incorporation of the chelator 1,10-phenanthroline at amino acid 10 of the helix-turn-helix motif. [(N-Acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP binds to a 22-base-pair DNA recognition site with Kobs = 1 x 10(8) M-1. In the presence of Cu(II) and reducing agent, [(N-acetyl-5-amino-1,10-phenanthroline)-Cys178]CAP cleaves DNA at four adjacent nucleotides on each DNA strand within the DNA recognition site. The DNA cleavage reaction has been demonstrated using 40-base-pair and 7164-base-pair DNA substrates. The DNA cleavage reaction is not inhibited by dam methylation of the DNA substrate. Such semisynthetic site-specific DNA cleavage agents have potential applications in chromosome mapping, cloning, and sequencing.

Hu, X.; Shima, S.

Angew. Chem. Int. Ed. 2021, 60, 13350-13357, 10.1002/anie.202100443

The reconstitution of [Mn]-hydrogenases using a series of MnI complexes is described. These complexes are designed to have an internal base or pro-base that may participate in metal–ligand cooperative catalysis or have no internal base or pro-base. Only MnI complexes with an internal base or pro-base are active for H2 activation; only [Mn]-hydrogenases incorporating such complexes are active for hydrogenase reactions. These results confirm the essential role of metal–ligand cooperation for H2 activation by the MnI complexes alone and by [Mn]-hydrogenases. Owing to the nature and position of the internal base or pro-base, the mode of metal–ligand cooperation in two active [Mn]-hydrogenases is different from that of the native [Fe]-hydrogenase. One [Mn]-hydrogenase has the highest specific activity of semi-synthetic [Mn]- and [Fe]-hydrogenases. This work demonstrates reconstitution of active artificial hydrogenases using synthetic complexes differing greatly from the native active site.

Koenigs, R.M.; Weissenborn, M.J.

ChemCatChem 2020, 10.1002/cctc.201901565

Bioinspired, synthetic porphyrin complexes are important catalysts in organic synthesis and play a pivotal role in efficient carbene transfer reactions. The advances in this research area stimulated recent, “chemo‐inspired” developments in biocatalysis. Today, both synthetic iron complexes and enzymes play an important role to conduct carbene transfer reactions. The advances and potential developments in both research areas are discussed in this concept article.

Hu, X.; Shima, S.

Nat. Chem. 2015, 7, 995-1002, 10.1038/Nchem.2382

[Fe]-Hydrogenase catalyses the reversible hydrogenation of a methenyltetrahydromethanopterin substrate, which is an intermediate step during the methanogenesis from CO2 and H2. The active site contains an iron-guanylylpyridinol cofactor, in which Fe2+ is coordinated by two CO ligands, as well as an acyl carbon atom and a pyridinyl nitrogen atom from a 3,4,5,6-substituted 2-pyridinol ligand. However, the mechanism of H2 activation by [Fe]-hydrogenase is unclear. Here we report the reconstitution of [Fe]-hydrogenase from an apoenzyme using two FeGP cofactor mimics to create semisynthetic enzymes. The small-molecule mimics reproduce the ligand environment of the active site, but are inactive towards H2 binding and activation on their own. We show that reconstituting the enzyme using a mimic that contains a 2-hydroxypyridine group restores activity, whereas an analogous enzyme with a 2-methoxypyridine complex was essentially inactive. These findings, together with density functional theory computations, support a mechanism in which the 2-hydroxy group is deprotonated before it serves as an internal base for heterolytic H2 cleavage.